Innate immune responses to bacterial infection mediate pathogen clearance and also modulate T cell responses. TNF, an inflammatory cytokine that is rapidly induced by microbial infection, is indispensable for clearance of many intracellular bacterial pathogens. The mechanism of TNF mediated protection, however, remains obscure. In this grant application we dissect early inflammatory events following murine infection with L. monocytogenes, focusing on TNF production by dendritic cells that are recruited to sites bacterial infection in an MCP-1/CCR2 dependent fashion. Our specific aims are: 1.) To identify precursors of TNF producing DCs in peripheral blood and to characterize stimuli which promote their recruitment and differentiation. MCP-1 production in spleen during L. monocytogenes infection will also be characterized. 2.) To investigate the mechanism of TNF mediated bacterial clearance. Specifically, we will test the hypothesis that in vivo TNF production is necessary for iNOS synthesis. The relative role of secreted versus membrane bound TNF in antimicrobial defense will also be tested. 3.) To characterize innate immune receptors and signaling pathways that induce in vivo TNF synthesis during L. monocytogenes infection. Specifically, we will characterize TNF production and iNOS induction in TLR-1, TLR-2, TLR-4, IRAK-M, TIRAP and MyD88 -/- mice. To facilitate these in vivo studies, we will generate GFP expressing TNF reporter mice and directly characterize cellular TNF induction during the course of L. monocytogenes infection. The experiments proposed in this grant application will provide an unprecedented view of in vivo innate immune recognition and antimicrobial defense, and will have important implications for the development of vaccines against common pathogens and potential agents of bioterror. [unreadable] [unreadable]